On the basis of the complete genome sequence of the budding yeast Saccharomyces cerevisiae, a computer-aided analysis was carried out of all members of the major facilitator superfamily (MFS), which typically consists of permeases with 12 transmembrane spans. Analysis of all 5885 predicted open reading frames identified 186 potential MFS proteins. Binary sequence comparison made it possible to cluster 149 of them into 23 families. Putative permease functions could be assigned to 12 families, the largest including sugar, amino acid, and multidrug transport. Phylogenetic clustering of proteins allowed us to predict a possible permease function for a total of 119 proteins. Multiple sequence alignments were made for all families, and evolutionary trees were constructed for families with at least four members. The latter resulted in the identification of 21 subclusters with presumably tightly related permease function. No functional clues were predicted for a total of 41 clustered or unclustered proteins.